Abstract
The radical centers detected in the reaction of metmyoglobin (MetMb) with hydrogen peroxide ($H_2O_2$) have been studied by using a spin trapping technique. A broad 5-line asymmetric electron spin resonance (ESR) spectrum, with $2A_{max}=4.07\;mT$ and $2A_{min}=2.97\;mT$, obtained after incubation of MetMb with $H_2O_2$ in the presence of a spin trap, 5,5-dimethyl pyrroline-N-oxide (DMPO) was gradually weakened with time and disappeared completely by 6 min after addition of guanidine-HCl (14 M). When a higher concentration (6 M) of the agent was added, the signal disappeared within 40 see and the DMPO/OH signal appeared immediately. Then, a new 8-line signal with similar intensities grew gradually and was fixed by 45 min, coexisting with the DMPO/OH signal. This new signal was found to be composite, consisting of two different radical species. One of the 6-line signals, with $a_N$ 1.49 mT and $a_H$ 0.988 mT, was assigned to the DMPO/phenoxyl radical adduct. The second 6-line signal with $a_N$ 1.55 mT and $a_H$ 2.22 mT was assigned to carbon-centered radical adduct. When 3,3,5,5-tetramethylpyrrolin-N-oxide (TMPO), was employed in the place of DMPO, another broad asymmetric 5-line signal was detected with $2A_{max}=3.99\;mT$ and $2A_{min}=3.04\;mT$, which is virtually identical to that obtained from the DMPO system The shape of the spectrum of the TMPO adduct changed drastically, with lapse of time resulting in a broad singlet after 40 min. The broad singlet was assigned to the porphyrin radical adduct. Incubation of globin with Fenton reagent in the presence of DMPO initially gave a DMPO/OH signal. Then, a new 12-line signal began to grow after one minute and fixed after 15 min. coexisting with the DMPO/OH signal, This 12-line signal was assigned to DMPO/phenoxyl with $a_N$ 1.47 mT, $a_{{\beta}H}$ 0.99 mT and $a_{{\gamma}H}$ 0.13 mT. A minor concentration of carbon-centered radical adduct was also detected. This radical composition is identical to that of guanidine HCl treated MetMb/DMPO/$H_2O_2$ system, indicating that the radical producing conditions are somehow common in both systems. Heme iron can be released by excess $H_2O_2$ in the MetMb/$H_2O_2$ system, providing for Fenton reagent. When MetMb was pretreated with tyrosine blocking agent, $KI_3$ the broad 5.line MetMb-derived signal was not detected in the MetMb/DMPO/$H_2O_2$ system, whereas no such effect was detected on such system of Hb in which the radical center was assigned to cysteine residue not tyrosine, indicating that tyrosine residue is a main radical center produced in the MetMb/$H_2O_2$ system Thus, the present data strongly support the previous indication that the apomyoglobin-derived radical center formed in the reaction of MetMb with $H_2O_2$ is a tyrosine residue.